Proteomic analyses today are very slow because each of the separation steps is slow. The separation speeds are limited by the materials. Presently, the workhorse of proteomics is 2D polyacrylamide gel electrophoresis (PAGE). These separations require many hours at low voltages to achieve efficient separations, and loss of protein on the gel reduces sensitivity, which is an especially severe problem for membrane proteins. We propose to address these problems with the use of colloidal crystals for 2D gel electrophoresis. Our goal is to achieve a 100-fold increase in speed of 2D separations of the most highly resolving gels, 20 cm in length, by using silica colloidal crystals of only 4 cm in length. In addition to the higher speed accrued from five-fold shorter travel distances, silica based materials allow more than an order of magnitude higher electric field. The combined miniaturization and higher field promises a combined 100-fold increase in speed along each dimension of the separation to give 2D separations in a few minutes rather than in many hours. The progress in the previous grant period addressed the materials science that demonstrated the promise, and the newly proposed work will carry the research from the materials stage to the separation stage. We propose to investigate transport in the media to understand how to maximize the overall speed of the separation. We propose to achieve on-chip integration of 2D separations: isoelectric focusing followed by sieving separation in the same colloidal crystal. We propose to improve the materials further by investigating inverse opal media, which have a larger free volume, yet the same pore size. We propose to study the sensitivity and dynamic range of fluorescence detection in differential gel electrophoresis for biomarker discovery. We propose to achieve mass spectrometric detection of protein from the colloidal crystals, investigating both MALDI and electrokinetic transfer of the protein from the colloidal crystal. The relevance of the research to public health is that a two order of magnitude increase in proteomic analyses would have an enormous impact on medical research, including determining mechanisms of drug action, the isolation of biomarkers to diagnose diseases, and an understanding of the onset of diseases for their prevention. PUBLIC HEALTH RELEVANCE The relevance of the research to public health is that a two order of magnitude increase in proteomic analyses would have an enormous impact on medical research, including determining mechanisms of drug action, the isolation of biomarkers to diagnose diseases, and an understanding of the onset of diseases for their prevention.